How is the process of development controlled? What is the circuity involved in a cell's decision to simply divide or to begin the process of differentiation? This process has been investigated in many organisms, but the process of sporulation in the soil bacteria, Bacillus subtilis is one of the best systems for studying these questions. Sporulation is induced when cells encounter nutrient deprivation. Thus the cell requires a mechanism to sense nutrient stress and an effector apparatus to convert sensory input into a suitable response. Since B. subtilis has several alternative responses to starvation, e.g., antibiotic and exoenzyme production, motility, competence and sporulation, etc., some of which have different nutritional and genetic requirements, the cell must also have ways to direct late growth development into these alternative pathways. Sporulation is also costly for a starved cell, in terms of resources required, and premature sporulation would be lethal for al cell, in a population sense. Therefore it would be expected that entry into this developmental process be controlled by fail/safe switches. Our laboratory has been studying SinR (formerly known as Sin), a 14kd DNA binding protein which is essential for competence, motility and autolysin production and is a repressor of sporulation and exoprotease synthesis. In its repressor function, SinR binds to the promoter regions of its target genes, preventing their transcription. Thus SinR plays the role of a developmental switch, so that when it is functional (ON) certain processes are activated (competence, motility and autolysin production) and others are repressed (sporulation and exoprotease production). When SinR is nonfunctional (OFF), the former genes are not activated and the latter are induced. The regulation of SinR function is therefore an important checkpoint in the cellular decisions regarding developmental fates. Recent experiments have shown that SinR function is regulated post-translationally by formation of a complex with the SinR protein that prevents SinR from binding to its target genes. Several questions regarding SinR's role in late growth development and its actual mechanism of action will be addressed in the course of this project. Does SinR act directly as a positive regulator of competence and motility regulon genes? How are the levels of SinI and SinR controlled to give the appropriate amount of functional SinR since their genetic determinants are in the same operon? Another area of importance concerns the actual mechanism of SinR action as a repressor and the modulation of this function by SinI. For example, how does SinR repress aprE when its binding site is more than 200 bp. upstream from the transcriptional start site? How does SinI interact with SinR at the molecular level to prevent its binding to target DNA sequence? Answers to these questions arising from our studies will provide valuable information on how cellular differentiation is controlled at the molecular level.